A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin–biased signaling

Gorvin, Caroline M; Babinsky, Valerie; Malinauskas, Tomas; Nissen, Peter H.; Schou, Anders; Hanyaloglu, Aylin C.; Siebold, Christian; Jones, E Y; Hannan, Fadil; Thakker, Rajesh V.

doi:10.1126/scisignal.aan3714

Cited by 34 publications

(46 citation statements)

References 56 publications

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“…Cells were lysed in Surefire lysis buffer, and AlphaScreen Surefire ERK assays (PerkinElmer, Waltham, MA, USA) measuring phosphorylated and total proteins performed, as described. (6,31) For studies with NPSP795, cells were incubated with either a 20% aqueous solution of 2-hydoxypropyl-β-cyclodextrin (vehicle) or NPSP795 for 4 hours prior to calcium treatment. The fluorescence signal in both assays was measured using the PheraStar FS microplate reader (BMG Labtech, Ortenberg, Germany).…”

Section: Intracellular Calcium Measurementsmentioning

confidence: 99%

“…The fluorescence signal in both assays was measured using the PheraStar FS microplate reader (BMG Labtech, Ortenberg, Germany). (6,31) Fold-change phosphorylated ERK (pERK) responses were expressed as a ratio of pERK to total ERK responses.…”

Section: Intracellular Calcium Measurementsmentioning

confidence: 99%

“…A utosomal dominant hypocalcemia (ADH) is a genetically heterogeneous disorder of extracellular calcium (Ca 2+ e ) homeostasis consisting of two reported variants: ADH type 1 (ADH1; OMIM #601198) is caused by germline gain-of-function mutations of the G-protein-coupled calcium-sensing receptor (CaSR), (1,2) and ADH type 2 (ADH2; OMIM #615361) is caused by germline gain-of-function mutations of G-protein subunit α-11 (Gα 11 ). (3)(4)(5)(6) The CaSR and Gα 11 proteins signal via multiple pathways, including intracellular calcium (Ca 2+ i ) mobilization and the ERK arm of the MAPK cascade to regulate PTH secretion and urinary calcium excretion. (7,8) ADH1 is the most common disease variant, with an estimated prevalence of 3.9 cases per 100,000, (9) and is characterized by hypocalcemia, increased circulating phosphate concentrations, inappropriately low or normal PTH concentrations, and a relative hypercalciuria with urinary calcium-to-creatinine ratios that are within or above the reference range.…”

Section: Introductionmentioning

confidence: 99%

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Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model

et al. 2020

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Calcilytics are calcium-sensing receptor (CaSR) antagonists that reduce the sensitivity of the CaSR to extracellular calcium. Calcilytics have the potential to treat autosomal dominant hypocalcemia type 1 (ADH1), which is caused by germline gain-of-function CaSR mutations and leads to symptomatic hypocalcemia, inappropriately low PTH concentrations, and hypercalciuria. To date, only one calcilytic compound, NPSP795, has been evaluated in patients with ADH1: Doses of up to 30 mg per patient have been shown to increase PTH concentrations, but did not significantly alter ionized blood calcium concentrations. The aim of this study was to further investigate NPSP795 for the treatment of ADH1 by undertaking in vitro and in vivo studies involving Nuf mice, which have hypocalcemia in association with a gain-of-function CaSR mutation, Leu723Gln. Treatment of HEK293 cells stably expressing the mutant Nuf (Gln723) CaSR with 20nM NPSP795 decreased extracellular Ca 2+-mediated intracellular calcium and phosphorylated ERK responses. An in vivo doseranging study was undertaken by administering a s.c. bolus of NPSP795 at doses ranging from 0 to 30 mg/kg to heterozygous (Casr +/ Nuf) and to homozygous (Casr Nuf/Nuf) mice, and measuring plasma PTH responses at 30 min postdose. NPSP795 significantly increased plasma PTH concentrations in a dose-dependent manner with the 30 mg/kg dose causing a maximal (≥10-fold) rise in PTH. To determine whether NPSP795 can rectify the hypocalcemia of Casr +/Nuf and Casr Nuf/Nuf mice, a submaximal dose (25 mg/kg) was administered, and plasma adjusted-calcium concentrations measured over a 6-hour period. NPSP795 significantly increased plasma adjusted-calcium in Casr +/Nuf mice from 1.87 AE 0.03 mmol/L to 2.16 AE 0.06 mmol/L, and in Casr Nuf/Nuf mice from 1.70 AE 0.03 mmol/L to 1.89 AE 0.05 mmol/L. Our findings show that NPSP795 elicits dose-dependent increases in PTH and ameliorates the hypocalcemia in an ADH1 mouse model. Thus, calcilytics such as NPSP795 represent a potential targeted therapy for ADH1.

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Section: Intracellular Calcium Measurementsmentioning

confidence: 99%

Section: Intracellular Calcium Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model

et al. 2020

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“…Figure 1A-B). SRE, NFAT and Fluo4-AM intracellular calcium mobilization assays were performed using methods previously described (37,38) in one of the cell lines -FlaC2 cells -to confirm a response to extracellular calcium stimulation (Supplemental Figure 1C), and therefore functionality of the CaSR signalling pathway. FlaC2 cells stably expressing WT (WT(Arg15)-AP2s2 FlaC2) or mutant (Leu15-AP2s2-FlaC2) AP2s2 proteins, were then generated and Ap2s1 +/L15 female mice, and snap frozen in liquid nitrogen, and subsequently stored at -80 o C. The outer renal capsule was removed to access the cortex, which was dissected and lysed in ice-cold lysis buffer, as described above, for co-IP analysis.…”

Section: Generation Of Ap2s1mentioning

confidence: 99%

Ap2s1mutation in mice causes familial hypocalciuric hypercalcemia type 3

Hannan

Stevenson

Bayliss

et al. 2020

Preprint

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Mutations of the adaptor protein-2 sigma subunit (AP2S1) gene which encodes AP2-sigma2, a component of the ubiquitous AP2 heterotetrameric complex involved in endosomal trafficking of the calcium-sensing receptor (CaSR), cause familial hypocalciuric hypercalcemia type 3 (FHH3). FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with marked hypercalcemia and occasional hypophosphatemia and osteomalacia. To further characterise the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcemia, hypermagnesemia, hypophosphatemia, and increased plasma concentrations of parathyroid hormone, fibroblast growth factor 23 and alkaline phosphatase activity, but normal pro-collagen type 1 N-terminal pro-peptide and 1,25 dihydroxyvitamin D. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. The AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2-sigma2 and the other AP2 subunits, and the CaSR. Cinacalcet, a CaSR allosteric activator, ameliorated the hypercalcemia and elevated PTH concentrations, but not the diminished AP2-sigma2-CaSR interaction. Thus, our studies have established a mouse model with a germline loss-of-function AP2S1 mutation that is representative for FHH3 in humans, and demonstrated that cinacalcet corrects the abnormalities of plasma calcium and PTH.

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“…The CaSR couples to two major signal transduction cascades that comprise the G q/11 -phospholipase C (PLC)-mediated generation of inositol 1,4,5-trisphosphate (IP 3 ), which induces a rapid rise in intracellular calcium (Ca 2+ i ) concentrations ( 7 ) and the mitogen-activated protein kinase (MAPK) cascade, such as the extracellular signal-regulated kinase 1/2 (ERK) pathway ( 8 ). CaSR-mediated activation of MAPK signalling can occur by coupling to either the G q/11 or G i/o pathways ( 9 ), and also by a G-protein-independent mechanism involving β-arrestin proteins ( 10 ).…”

Section: Introductionmentioning

confidence: 99%

Calcium-sensing receptor residues with loss- and gain-of-function mutations are located in regions of conformational change and cause signalling bias

Gorvin

Frost

Malinauskas

et al. 2018

Human Molecular Genetics

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The calcium-sensing receptor (CaSR) is a homodimeric G-protein-coupled receptor that signals via intracellular calcium (Ca2+i) mobilisation and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) to regulate extracellular calcium (Ca2+e) homeostasis. The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss- and gain-of-function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five ‘disease-switch’ residues (Gln27, Asn178, Ser657, Ser820 and Thr828) that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease-switch residue mutations to commonly display signalling bias. For example, two FHH-associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH-associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease-switch residues together with four reported disease-switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease-switch residues are located at sites critical for CaSR activation and play a role in mediating signalling bias

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A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin–biased signaling

Cited by 34 publications

References 56 publications

Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model

Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model

Ap2s1mutation in mice causes familial hypocalciuric hypercalcemia type 3

Calcium-sensing receptor residues with loss- and gain-of-function mutations are located in regions of conformational change and cause signalling bias

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